Project Summary/Abstract ADARs are RNA editing enzymes that convert adenosine to inosine in cellular and viral double- stranded RNA (dsRNA). One function of ADARs is to target codons in mRNA to allow multiple protein isoforms from the information in a single gene. Codon editing is found in many neuronally important mRNAs, and aberrant levels of editing have been linked to neurological disease. Fundamental to determining how ADARs contribute to human health, and sometimes disease, is a complete understanding of their catalytic mechanism. Experiments are proposed to determine the amino acids in the human ADAR2 catalytic domain that control its catalytic efficiency and lead to targeting of precise adenosines. A screen has identified mutations that affect these proceses, and these wil be characterized using a variety of biochemical assays. Despite its importance, editing in codons is rare, and there are far more inosines in noncoding RNA sequences. Yet, the function of inosines in noncoding sequences is unclear. Since ADARs target any dsRNA sequence, one posibility is that they affect dsRNA-mediated gene silencing pathways. High- throughput sequencing will be performed to compare the small RNAs of wildtype C. elegans with those in strains lacking ADARs. The focus will be on small RNAs that are processed from a dsRNA precursor, namely, microRNAs and endogenous siRNAs. Altered levels of small RNAs as well as their editing sites will be tabulated, and the latter will be distinguished from sequencing errors by their absence in animals lacking ADAR editing. Effects of ADARs on small RNAs will be correlated with predicted changes in mRNA levels using microarray analyses. While C. elegans lacking ADARs are viable, they have chemotaxis defects, and it is anticipated they have other subtle defects that have not been recognized. After validation of observations made with bioinformatics studies, phenotypes suggested by molecular defects will be tested. PUBLIC HEALTH RELEVANCE: Project Narrative ADARs are RNA editing enzymes that are essential for human life. They have been linked to longevity, and aberrant levels of editing have been associated with neurological disease and cancer. The proposed work will enhance our understanding of how ADARs contribute to human health, as well as disease, by advancing our understanding of the enzyme's catalytic mechanism and its substrates.